NO167460B - PROCEDURE FOR CLEANING AN ANTIBIOTIC CONTENT. - Google Patents

Description

The present invention relates to another method

purification of an antibiotic-containing material.

The following designations will be used in what follows:

Antibiotic (BMY-28100):

(E)-isomer (BMY-28167): antibiotic sodium salt: sodium 7-|D-2-amino-2-(4-hydroxyphenyl)acetamido|-3-((Z)-1-propenyl)-cef-3-em-4-carboxylate (E)- isomeric sodium salt: sodium 7-|D-2-amino-2-(4-hydroxyphenyl)acetamido|-3-((E)-1-propenyl)cef-3-em-4-carboxylate

(z)-isomer imidazolidinone derivative sodium salt:

where R and R<1> are alkyl, alicyclyl, aryl or aralkyl groups of the ketone of the formula RCR^" or R and R<1> are joined together to form a cyclic ketone having a molecular weight of less than 200 ( E)-isomerimidazolidinone derivative sodium salt:

where R and R^ have the meaning indicated above.

When R and R^ are methyl groups, the imidazolidinone derivatives shown above are sometimes called acetonides.

Antibiotic BMY-28100 and its (E)-isomer BMY-28167 are described in US Patent 4,520,022. These substances are produced synthetically by methods which involve the formation of a 3-(l-propenyl) group via the reaction of acetaldehyde with phosphoranyl intermediates, which are prepared from 3-halomethylcephalosporin starting materials. This type of synthesis is known as a Wittig synthesis, and in the present case mixtures of cis- and trans-isomers are formed around the propenyl double bond. The cis isomer is called the (Z) isomer, and the trans isomer is called the (E) isomer. The (Z)-isomer is the preferred form as this has better Gram-negative antibacterial activity compared to the (E)-isomer. For pharmaceutical purposes, it is desirable to use the (Z)-isomer in a form that is largely free of the (E)-isomer for

to ensure maximum antibiotic effect. With the expression "largely free" is meant a content of the (E)-isomer that does not exceed 2% by weight.

The problem that the technique is thus faced with is to recover BMY-28100 from mixtures with the (E) isomer. The methods 8 and 9 listed in columns 20 and 21 of the above-mentioned US patent are preparative liquid chromatography methods for recovering the antibiotic in a form substantially free of the (E)-isomer from a material containing the antibiotic and the (E)-isomer in mixture. Liquid chromatography is not suitable for large-scale production of clinical and commercial quantities of an antibiotic such as this. Other conventional physical methods for the separation of about an olefinic double bond geometric isomers, such as fractional recrystallization and selective absorption methods, have not been successful with BMY-28100-containing mixtures.

Imidazolidinone derivatives of the type produced according to the invention have previously been prepared with cephalosporin and penicillin antibiotics containing an -amino- -phenylacetamide substituent in the 7-position. It can e.g. reference is made to US Patent 3,198,804 and to G. A. Hardcastle et al., J. Org. Chem., Vol. 31, 1966, p. 897, which describes ampicillin, derivatives which serve as prodrugs and are administered for therapeutic purposes. The antibiotic itself is released in the body. US patent 3,994,884 relates to corresponding derivatives of cephalosporins containing a vinyl group in the 3-position, and GB patent 1,503,310 relates to such derivatives of cefatrizine. Furthermore, US patent 4,026,688 relates to oxazolidinone derivatives of cefatrizine with various aromatic aldehydes. However, no use of such derivatives is known to separate mixtures of cephalosporins where there is geometric isometry around a double bond.

The question of controlling the ratio between the cis- and trans-isomers formed in the Wittig reaction with aldehydes by influencing the course of the reaction by means of solvents and various additives has been investigated by H. 0. House et al., Journal of Organic Chemistry, Vol. 29, 1964, pp. 3327-3333.

The method according to the invention is characterized by the fact that the material, which contains a crystalline solvate of the antibiotic 7-ID-2-amino-2-(4-hydroxyphenyl)acetamido|-3-| (Z)-1-propenylIcef-3-em-4-carboxylic acid in a mixture with its (E)-isomer is reacted with sodium 2-ethyl hexanoate, in the presence of acetone, in a liquid medium which, in addition to acetone, can comprise a reaction-inert solvent, preferably methanol, at a temperature of 35-55°C, under such conditions that the antibiotic imidazolidinone derivative sodium salt and the (E)-isomerimidazolidinone sodium salt are formed, that imidazolidinone derivatives formed in steps are suspended in a liquid alkanol, which can comprise acetone and wherein the (E)-isomerimidazolidinone derivative sodium salt is soluble and the antibiotic imidazolidinone derivative sodium salt is largely insoluble, to form a suspension of the antibiotic derivative in a solution of the (E)-isomer derivative, and that the insoluble antibiotic derivative is recovered from the suspension in a form which is largely free of the (E)-isomer derivative, and that, if desired, the recovered antibiotic derivative is converted into said antibiotic in a known manner.

By the method according to the present invention, a 3-|(Z)-1-propenyl|cephalosporinimidazolidinone carboxylic acid derivative with the formula is prepared

where R and R"'" are organic groups from an alkyl-,

O

il in alicyclyl, aryl or aralkyl ketone of the formula RCR and with a molecular weight of not more than 200, a pharmaceutically acceptable metal or other salt thereof, or a crystalline solvate of the acid or salt with a pharmaceutically acceptable liquid. R and R^" can be linked together to form an alicyclic ketone, such as cyclohexanone, cyclohexenone or cyclopentanone. Preferred compounds are those where one of the groups R and R is alkyl of up to 4 carbon atoms, or where R and R<1> together with the carbon atom to which they are attached form a 6-membered ring The most preferred compounds are those where both R and R"*" are methyl.

These compounds are of particular interest as intermediate products in a purification method for the preparation of the antibiotic with cis- or (Z) configuration and mostly without content of

The (E) isomer. Methods for the preparation of the antibiotic usually lead to mixtures, which contain both (Z)- and (E)-

the isomers, while it is the (Z) isomer that is preferred for pharmaceutical purposes. According to the present invention, it has been shown that the imidazolidinone derivative sodium salts of the antibiotic are practically insoluble in various organic solvents, while the corresponding (E)-isomerimidazolidinone derivative sodium salts are soluble. This makes it possible to separate the desired (Z) configuration of the antibiotic, as the crystalline sodium salt of the derivative shown above, from which the antibiotic itself can be recovered or released.

These substances can be prepared as a pharmaceutically acceptable metal or amine salt. They are often isolated as crystalline solvates, where the crystal contains a certain amount of the liquid from which they are crystallized. By solvates is meant a crystalline compound that contains a certain amount of solvent in the crystal lattice, usually the crystal structure that can be characterized by X-ray powder diffraction spectrometry. They are stable solids at room temperature. The preferred compound according to the invention corresponds to the above formula, where R and R are methyl groups, and substances are isolated as sodium salt monohydrate, which is a crystalline solid.

The above-mentioned substances are prepared from the antibiotic BMY-2810 0 by converting this to an alkali metal salt in the presence of a ketone of the above type. The products are isolated as alkali metal salts. The pharmaceutically acceptable metal and amine salts are useful as dry pharmaceutical preparations because of their stability in the solid state. Those that are water-soluble are particularly suitable for the preparation of parenteral dosage forms. They are converted by hydrolysis or enzymatically in the body to the antibiotic BMY-2 810 0. Hydrolysis occurs under acidic pH conditions, such as at pH 3-7, which are found in various mammalian tissues or organs. Strong aqueous acid (pH below 1) converts the metal and amine salts into the water-insoluble acid form of the oxazolidinone products according to the present invention.

The drawing shows the IR spectrum of a product obtained according to the present invention, prepared according to example 1. For use in the measurement, the crystalline sample was formed as a pill using crystalline potassium bromide as carrier.

The method of the present invention can be applied to BMY-28100 antibiotic materials, which contain from 2 to 65% by weight (E)-isomer. The material containing 2% or less of the (E) isomer is considered substantially free of the (E) isomer for pharmaceutical purposes. It is an aim of the present invention to produce a method for producing antibiotic BMY-28100 preparations, which are largely free of the (E) isomer. Another purpose is to produce the antibiotic BMY-28100 prodrug derivatives, which are particularly suitable for pharmaceutical dosage preparations.

In the method according to the present invention, an antibiotic BMY-2810O material, which contains from 2 to 65% by weight (E)-isomer, is first reacted with a sodium salt-forming reactant, i.e. sodium 2-ethyl hexanoate in the presence of a ketone reactant

0

RCR^ -in the form of acetone. The sodium salt of an acid, which is a weaker acid than the antibiotic or its (E)-isomer, can otherwise be used as the sodium salt-forming reactant. The reaction is preferably carried out in solution, and for the first step it is desirable to choose solvents and salt-forming reactants which result in the formation of a solution at the beginning of the reaction or as it progresses. As the imidazolidinone derivatives are formed, precipitation of the antibiotic imidazolidinone derivative sodium salt occurs as a result of its lower solubility in relation to the (E)-isomerimidazolidinone derivative sodium salt. After the reaction is finished, the antibiotic imidazolidinone derivative sodium salt is recovered by filtration or centrifugation. The resulting product is in itself useful for pharmaceutical purposes, or can be converted into the antibiotic itself which is largely free of the (E) isomer.

When carrying out the method according to the invention, as mentioned, sodium 2-ethylhexanoate is used as a salt-forming reactant, but other reactants, such as sodium acetate or the sodium salt of other carboxylic acids, can be used. A reaction-inert organic solvent, such as a liquid alkanol, is preferably used as solvent in combination with the ketone/acetone which is used as the imidazolidinone-forming reactant. A minimal amount of water can also be used to make the dissolution easier. The preferred solvents are mixtures of methanol and acetone. The acetonides of BMY-28100 and BMY-28167 are very soluble in methanol and largely insoluble in acetone. As a result, mixtures of these two solvents are used to achieve the desired solubility difference. Mixtures of methanol and acetone, containing from 20 to 50% by volume of methanol, are suitable. The antibiotic 2,2-dimethylimidazolidinone derivative sodium salt is insoluble in the methanol-acetone mixture at room temperature or lower, while the 2,2-imidazolidinone derivative sodium salt of the (E) isomer is soluble. This difference in solubility provides a basis for separating these otherwise very similar substances.

The reaction is preferably carried out at temperatures of 40-55°C, which gives a more manageable crystallization of the product.

When alkyl, aryl or aralkyl ketones are used which are not suitable solvents for the antibiotic and its (E)-isomer, the ketone can be used dissolved in an organic solvent and in an amount that is approx. chemically equivalent to the antibiotic and

The (E)-isomer thereof. In this case, a solvent is selected in which the antibiotic imidazolidinone derivative sodium salt, which

is formed from this ketone, is insoluble, while (E)-isomerimidazo-lidinone derivative sodium salt is soluble. Testing different combinations of ketones, salt-forming reactants and solvents to achieve this goal belongs to the skills of a laboratory chemist.

The method according to the present invention can be used on antibiotic BMY-28100 preparations when solvate forms of the antibiotic and its (E) isomer are available. The method according to the above-mentioned US patent document 4,520,022 gives e.g. the crystalline monohydrate, which is a suitable form for treatment by the method according to the present invention. Another suitable form is the dimethylformamide solvate, which is formed by the process of US patent application 759,805.

Pharmaceutically acceptable metal and amine salts are such salts which are stable under ambient conditions and in which the cation does not contribute significantly to the salt's toxicity or biological activity. Suitable metal salts include sodium, potassium, calcium, zinc and magnesium salts. The sodium or potassium salts are highly water soluble and are preferred. Amine salts which are prepared from amines, such as e.g. used in conjunction with benzylpenicillin and which are capable of forming stable salts with the acidic carboxyl group, include trialkylamines such as triethylamine, procaine, dibenzylamine, N-benzyl- -phenethylamine, 1-ephenamine, N,N<1->dibenzylethylenediamine , dihydroabiethylamine, N-ethylpiperidine, benzylamine and dicyclohexylamine. Some of these amine salts are insoluble in water.

The utility of sodium 7-£2,2-dimethyl-4-(4-hydroxy-phenyl)-5-oxo-1-imidazolidinyl]-3-£( Z)-1-propenyl]cef-3-em-4 -carboxylate as a dosage form in the form of a solution for parenteral injection was demonstrated by preparing an aqueous solution containing 250 mg/ml thereof and measuring the concentration of antibiotic in the solution at room temperature for a storage period of several hours. It turned out that the solution at a pH value of approx. 7.2 lost 10% strength of approx. 2 hours. This stability is far better than the stability of the sodium salt of the antibiotic itself, which showed a pH value of 8.2 and which lost 10% of its potency in 30 minutes. While a 10% loss in potency is acceptable, an adequate pot life of at least 2 hours is required for an antibiotic solution prepared on demand.

The stability of the substance in solid form was demonstrated by storing samples of it at different temperatures for different periods of time and measuring the purity of the sample at different intervals. The results appear in the following table:

A further advantage of the method according to the invention is that the antibiotic which is recovered by hydrolysis of the imidazolidinone derivative sodium salt contains a smaller amount of impurities.

Example 1 - Imidazolidinone derivatives from acetone

A solid material contained 83% of the crystalline monohydrate of antibiotic BMY-2 810 0 and 17% of the (E) isomer thereof (BMY-28167), and 102 g of this material was mixed with 2 L of acetone, after which the slurry was heated with stirring to 40°C. With continued stirring, 83.1 g (0.5 M) of sodium 2-ethyl hexanoate in powder form was added, followed by 1.2 L of methanol. During 5 minutes of continued stirring at 40°C, a clear solution was formed. After 15 minutes, the sodium salt of the acetonimidazolidinone derivative of the antibiotic, namely sodium 7-|2,2-(dimethyl-4-(4-hydroxyphenyl)-5-oxo-1-imidazolidinyl|-3-|1-(Z)- propenyl-|-cef-3-em-4-carboxylate to crystallize. After 1 hour the mixture became very thick and 1 L of a 1:1 volume mixture of methanol and acetone was added to facilitate stirring. After stirring for an additional 1 hour at 40°C, the mixture was cooled at 15-20°C with stirring, and the insoluble product was then isolated by vacuum filtration.The moist filter cake was suspended in a mixture of 2 L of acetone and 0.5 L of methanol and shaken for 1 h at 20° C. The product was collected by filtration, washed on the filter with 0.2 L of a 2:8 volume mixture of methanol and acetone and then with acetone and transferred to a vacuum desiccator and dried. Another yield was obtained by concentrating the filtrate to 0.5 L and mixing the concentrate with 0.5 L of acetone This material was collected by filtration, resuspended in 1 L of a 1 :5 volume mixture of methanol and acetone, collected and dried, yielding an additional 22.1 g of product equal in quality to the first yield. A total of 86.1 g (93%) was obtained.

Analytical data:

A. structure:

The product obtained as a crystalline hydrate.

Elemental analysis:

The IR spectrum appears in the drawing. The UV spectrum showed the following data: max = 222nm (E = 14423) NMR: Chemical shifts (relative to trimethylsilylpropane sulfonic acid 0.ppm in DjO with elimination of water)

Other ketones, such as cyclohexanone, methyl ethyl ketone, cyclopentanone, methyl vinyl ketone and methyl isobutyl ketone can be used instead of acetone if necessary.

Example 2 - Hydrolysis of imidazolidinone derivatives

to the formation of antibiotics

30 g of the imidazolidinone derivative prepared in Example 1 was added as dry powder to 175 ml of 40°C warm water in a reaction vessel equipped with a stirrer, thermometer, drop funnel and pH electrode. The pH value of the reaction mixture rose as the acetone derivative went into solution and was kept in the range from pH 5.8 to 6.0 by means of the dropwise addition of 1N HCl. After 15 minutes, a second 30 g portion of the imidazolidinone derivative was added while maintaining the pH value of 5.8-6.0. A final portion, 26 g, of the imidazolidinone derivative was added in the same way approx. 15 minutes after the second portion. The pH value was monitored for a period of 4.5 hours and maintained during this period at 5.8-6.0 by addition of IN HCl. The mixture was then cooled to approx. 0°C, and the pH was adjusted to 4.1 by addition of 1N HCl. After 20 minutes, the product was collected by filtration, the filter cake was washed with 100 ml of ice-cold water and dried in a vacuum. The yield was 62.4 g (83.6%). This material was shown by PMR to contain 1.5% by weight of the (E) isomer and by liquid chromatography to be composed of 94.9% by weight of the desired antibiotic (Z) isomer and of 1.78% by weight of (E ) isomer.

The following system is suitable for liquid chromatography analysis of the raw material from Example 1 and of the product from Example 2 to determine the ratio between the (Z) and (E) components (BMY-28100/BMY-28167).

Example 3 - Conversion of imidazolidinone derivative-

sodium salt with sodium bisulfite

12 5 mg of the product from example 1 was dissolved in 1 ml of water at 45-50°C. While stirring this solution, 62.5 g (approx. 2.5 molar equivalents) of sodium bisulphite were added over the course of 20 minutes. In this step, it is appropriate to add seed crystals of BMY-28100 monohydrate, if such crystals are available, but this is not essential. Heating and stirring continued for half an hour. The mixture was cooled to room temperature and stirred for another half hour, after which the product was collected. The yield was approx. 60% BMY-2 810 0 monohydrate with a content of 1.8% of the (E) isomer thereof.

Example 4 - Acetonimidazolidinone derivative from

BMY- 2810 0- dimethylformamide solvate

Materials: 4.62 g (0.0082 M) BMY-28100. 1.5 DMF

3.32 g (0.02 M) sodium 2-ethyl hexanoate 100 ml acetone

40 ml of methanol

0.18 mL (0.1 M) of water

A slurry of BMY-28100. 1.5 DMF in the mixture of methanol and acetone was stirred at room temperature while adding sodium 2-ethyl hexanoate and then water. The mixture was heated to 40°C with continued stirring. A clear solution was formed within 5 minutes. About. 10 minutes later a precipitate began to form. The mixture was still stirred at 40°C for 2

1/2 hour and then cooled to approx. 18°C, and the product was collected. The product was largely identical to the product produced in example 1.

Claims (6)

1. Method for purifying an antibiotic-containing material, characterized in that (1) the material, which contains a crystalline solvate of the antibiotic 7-Q)-2-amino-2-(4-hydroxyphenyl)acetamido]-3-DZ)-1 -propenyl]cef-3-em-4-carboxylic acid in a mixture with the (E)-isomer thereof, is reacted with sodium 2-ethyl hexanoate, in the presence of acetone, in a liquid medium which, in addition to acetone, may comprise a the reaction inert solvent, preferably methanol, at a temperature of 35-55°C, under such conditions that the antibiotic imidazolidinone derivative sodium salt and the (E)-isomer imidazolidinone sodium salt are formed, (2) that the imidazolidinone derivatives formed in step (1) are suspended in a liquid alkanol, which may comprise acetone and in which the (E)-isomer imidazolidinone derivative sodium salt is soluble and the antibiotic imidazolidinone derivative sodium salt is largely insoluble, to form a suspension of the antibiotic derivative in a solution of the (E)-isomer derivative, and (3) that the insoluble antibiotics m derivative is recovered from the suspension in a form that is largely free of the (E)-isomer derivative, and that, if desired, the recovered antibiotic derivative is converted into said antibiotic in a known manner. 2. Method in accordance with claim 1, characterized in that the reaction is carried out at a temperature of 40-55°C. 3. Method in accordance with claim 1, characterized in that the solvent in step (2) comprises acetone. 4. Method in accordance with claim 1, characterized in that the solvent in step (2) is a mixture of methanol and acetone. 5. Method in accordance with claim 1, characterized in that the solvate is the hydrate. 6. Method in accordance with claim 1, characterized in that the solvate is the dimethylformamide solvate.